The need to introduce targeted modifications in plant genomes, e.g. to provide plants with agronomically useful traits such as herbicide tolerance, including the control over the location of integration of foreign DNA in plants has become increasingly important. Several methods have been developed in an effort to meet this need (for a review see Kumar and Fladung, 2001, Trends in Plant Science, 6, pp 155-159), which mostly rely on the initial introduction of a double stranded DNA break at the targeted location via expression of a double strand break inducing (DSBI) enzyme.
Activation of the target locus and/or repair or donor DNA through the induction of double stranded DNA breaks (DSB) via rare-cutting endonucleases, such as I-SceI has been shown to increase the frequency of homologous recombination by several orders of magnitude. (Puchta et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, pp 5055-5060; Chilton and Que, Plant Physiol., 2003; D'Halluin et al. 2008 Plant Biotechnol. J. 6, 93-102).
WO96/14408 describes an isolated DNA encoding the enzyme I-SceI. This DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.
WO00/46386 describes methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell through an I-SceI induced double strand break. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof. Further disclosed are chimeric restriction endonucleases.
WO 2005/049842 describes methods and means to improve targeted DNA insertion in plants using rare-cleaving “double stranded break” inducing (DSBI) enzymes, as well as improved I-SceI encoding nucleotide sequences.
WO2006/105946 describes a method for the exact exchange in plant cells and plants of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print and without resorting to in vitro culture during the removal step, employing the therein described method for the removal of a selected DNA by microspore specific expression of a DSBI rare-cleaving endonuclease.
WO2008/037436 describe variants of the methods and means of WO2006/105946 wherein the removal step of a selected DNA fragment induced by a double stranded break inducing rare cleaving endonuclease is under control of a germline-specific promoter. Other embodiments of the method relied on non-homologous endjoining at one end of the repair DNA and homologous recombination at the other end. WO08/148559 describes variants of the methods of WO2008/037436, i.e. methods for the exact exchange in eukaryotic cells, such as plant cells, of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print employing a method for the removal of a selected DNA flanked by two nucleotide sequences in direct repeats.
WO 2003/004659 discloses recombination systems and a method for removing nucleic acid sequences from the chromosomal DNA of eukaryotic organisms. The invention also relates to transgenic organisms (preferably plants), containing said systems or produced by said method.
WO 2006/032426 discloses improved recombination systems and methods for eliminating maker sequences from the genome of plants. Particularly the invention is based on use of an expression cassette comprising the parsley ubiquitin promoter, and operably linked thereto a nucleic acid sequence coding for a sequence specific DNA-endonuclease.
U.S. provisional application 61/493,579 and EP11004570.5 describe methods and means to modify in a targeted manner the genome of a cotton plant using a double stranded DNA break inducing enzyme and embryogenic callus.
In addition, methods have been described which allow the design of rare cleaving endonucleases to alter substrate or sequence-specificity of the enzymes, thus allowing to induce a double stranded break at a locus of interest without being dependent on the presence of a recognition site for any of the natural rare-cleaving endonucleases. Briefly, chimeric restriction enzymes can be prepared using hybrids between a zinc-finger domain designed to recognize a specific nucleotide sequence and the non-specific DNA-cleavage domain from a natural restriction enzyme, such as Fold. Such methods have been described e.g. in WO 03/080809, WO94/18313 or WO95/09233 and in Isalan et al., 2001, Nature Biotechnology 19, 656-660; Liu et al. 1997, Proc. Natl. Acad. Sci. USA 94, 5525-5530). Another way of producing custom-made meganucleases, by selection from a library of variants, is described in WO2004/067736. Custom made meganucleases or redesigned meganucleases with altered sequence specificity and DNA-binding affinity may also be obtained through rational design as described in WO2007/047859.
WO2007/049095 describes “LADGLIDADG” homing endonuclease variants having mutations in two separate subdomains, each binding a distinct part of a modified DNA target half site, such that the endonuclease variant is able to cleave a chimeric DNA target sequence comprising the nucleotides bound by each subdomain.
WO2007/049156 and WO2007/093836 describe I-CreI homing endonuclease variants having novel cleavage specificity and uses thereof.
WO2007/047859 describes rationally designed meganucleases with altered sequence specificity and DNA binding affinity.
WO11/064736 describes optimized endonucleases, as well as methods of targeted integration, targeted deletion or targeted mutation of polynucleotides using optimized endonucleases. WO11/064750 describes chimeric endonucleases, comprising an endonuclease and a heterologous DNA binding domain comprising one or more Zn2C6 zinc fingers, as well as methods of targeted integration, targeted deletion or targeted mutation of polynucleotides using chimeric endonucleases and WO11/064751 describes chimeric endonucleases, comprising an endonuclease and a heterologous DNA binding domain, as well as methods of targeted integration, targeted deletion or targeted mutation of polynucleotides using chimeric endonucleases.
PCT/EP11/002894 and PCT/EP11/002895 describe methods and means to modify in a targeted manner the plant genome of transgenic plants comprising chimeric genes wherein the chimeric genes have a DNA element commonly used in plant molecular biology, as well as re-designed meganucleases to cleave such an element commonly used in plant molecular biology.
WO 2009/006297 discloses methods and compositions for altering the genome of a monocot plant cell, and a monocot plant, involving the use of a double-stranded break inducing agent to alter a monocot plant or plant cell genomic sequence comprising a recognition sequence for the double-stranded break inducing agent.
Gao et al. 2010, The Plant Journal 61, p 176-187 describe heritable targeted mutagenesis in maize using a re-designed endonuclease.
However, in order to efficiently make combinations of agronomically useful traits without having to resort to elaborate breeding schemes or to test large numbers of single events, there thus still remains a need for functional re-designed meganucleases which can recognize a recognition site in close proximity to an already existing elite event, and uses thereof in order to make stacks of genes conferring agronomically favorable properties at a single genetic locus.
One of such agronomically useful traits is tolerance to herbicides, such as HPPD-inhibitor herbicides. HPPD (hydroxyphenylpyruvate dioxygenase) proteins are enzymes which catalyse the reaction in which para-hydroxyphenylpyruvate (abbreviated herein as HPP), a tyrosine degradation product, is transformed into homogentisate (abbreviated herein as HG), the precursor in plants of tocopherol and plastoquinone (Crouch N. P. et al. (1997) Tetrahedron, 53, 20, 6993-7010, Fritze et al., (2004), Plant Physiology 134:1388-1400). Tocopherol acts as a membrane-associated antioxidant. Plastoquinone, firstly acts as an electron carrier between photosystem II (PSII) and the cytochrome b6/f complex and secondly, is a redox cofactor for phytoene desaturase, which is involved in the biosynthesis of carotenoids.
Up to now, more than 700 nucleic acid sequences from various organisms present in NCBI database were annotated as coding for a putative protein having an HPPD domain. Several HPPD proteins and their primary sequences have been described in the state of the art, in particular the HPPDs of bacteria such as Pseudomonas (Rüetschi et al., Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), of plants such as Arabidopsis (WO 96/38567, Genebank AF047834), carrot (WO 96/38567, Genebank 87257), Avena sativa (WO 02/046387), wheat (WO 02/046387), Brachiaria platyphylla (WO 02/046387), Cenchrus echinatus (WO 02/046387), Lolium rigidum (WO 02/046387), Festuca arundinacea (WO 02/046387), Setaria faberi (WO 02/046387), Eleusine indica (WO 02/046387), Sorghum (WO 02/046387), Coccicoides (Genebank COITRP), of Coptis japonica (WO 06/132270), Chlamydomonas reinhardtii (ES 2275365), or of mammals such as mouse or pig.
Inhibition of HPPD leads to uncoupling of photosynthesis, deficiency in accessory light-harvesting pigments and, most importantly, to destruction of chlorophyll by UV-radiation and reactive oxygen species (bleaching) due to the lack of photo protection normally provided by carotenoids (Norris et al. (1995), Plant Cell 7: 2139-2149). Bleaching of photosynthetically active tissues leads to growth inhibition and plant death.
Some molecules which inhibit HPPD, and which bind specifically to the enzyme in order to inhibit transformation of the HPP into homogentisate, have proven to be very effective selective herbicides. At present, most commercially available HPPD inhibitor herbicides belong to one of these three chemical families:                1) the triketones, e.g. sulcotrione, mesotrione; tembotrione; tefuryltrione; bicyclopyrone; benzobicyclon        2) the isoxazoles, e.g. isoxaflutole, or corresponding diketonitriles. In plants, isoxazoles such as isoxaflutole are rapidly converted into diketonitriles, which exhibit the HPPD inhibitor property; and        3) the pyrazolinones, e.g. topramezone, pyrasulfotole and pyrazoxyfen.        
These HPPD-inhibiting herbicides can be used against grass and/or broad leaf weeds in crop plants that display metabolic tolerance, such as maize (Zea mays) in which they are rapidly degraded (Schulz et al., 1993; Mitchell et al., 2001; Garcia et al., 2000; Pallett et al., 2001). In order to extend the scope of these HPPD-inhibiting herbicides, several efforts have been developed in order to confer to plants, particularly plants without or with an underperforming metabolic tolerance, a tolerance level acceptable under agronomic field conditions.
In that context, it has first been demonstrated that the mere overexpression of a native HPPD enzyme in transformed sensitive plants does provide an effective tolerance to HPPD inhibitors to the transformed plants (WO96/38567).
Another strategy was to mutate the HPPD in order to obtain a target enzyme which, while retaining its properties of catalysing the transformation of HPP into homogentisate, is less sensitive to HPPD inhibitors than is the native HPPD before mutation.
This strategy has been successfully applied for the production of plants tolerant to HPPD-inhibitors, by transforming plants with a gene encoding an HPPD enzyme mutated at one or more positions in its C-terminal part (WO 99/24585). Among the useful mutations in the C-terminal part of HPPD enzymes which can confer tolerance to HPPD-inhibitors, certain mutations were shown to provide increased tolerance to certain diketonitrile herbicides, for example the mutations Pro215Leu, Gly336Glu, Gly336Ile, and Gly336Trp (positions of the mutated amino acid are indicated with reference to the Pseudomonas HPPD).
More recently, it has been shown in patent application WO 2009/144079 that certain specific amino acid substitutions at position 336 of the HPPD provide tolerance to certain HPPD inhibitor herbicides in vitro.
US 2010/0197503 also indicates a number of mutations at different positions within or close to the active site of the HPPD taken from Avena sativa and examines some of these mutated HPPD enzymes for their inhibition by certain HPPD inhibitors such as sulcotrione.
Despite these successes obtained for the development of plants showing tolerance to some HPPD inhibitors herbicides described above, it is still desirable to develop and/or improve the tolerance of specific plants such as cotton, to more, newer or to several different HPPD inhibitors, particularly HPPD inhibitors belonging to the classes of the triketones (e.g. sulcotrione, mesotrione, tembotrione, tefuryltrione, bicyclopyrone and benzobicyclon), the pyrazolinones (e.g., topramezone, pyrasulfotole and pyrazoxifen) and the isoxazoles (e.g. isoxaflutole) or corresponding diketonitriles. This problem is solved as herein after described in the different embodiments, examples and claims.
These and other problems are solved as described hereinafter in the different detailed embodiments of the invention, as well as in the claims.